When an electrical field was applied across a medium it caused charged particles to migrate within the medium as a function of the composition of the medium, the parameters of the electrical source and of the characteristics of the particles themselves. The composition of the medium and the parameters of the electrical source were reproducible factors so that the composition of particles which have migrated within the medium was accurately reflected in their relative positions after migration. Such phenomenon has been employed as the basis for electrophoresis.
Various electrophoretic systems have been used for separating complex mixtures of molecules. Both slab and columnar electrophoretic apparatus have been employed in conjunction with conducting media such as paper, agarose and polymers. A medium was positioned between two tanks which carried electrolytic solutions, and an electrical potential difference was applied across the tanks to produce migration of particles within the medium according to charge or size.
Identification of the different cell proteins by electrophoresis has become an analytic procedure of major significance in biological diagnostics.
Among the prior electrophoretic systems, separation of proteins according to differences in size has been achieved by employing a medium of sodium dodecyl sulfate polyacrylamide gel which has the potential to resolve 10.sup.2 different types of proteins out of the estimated 5.times.10.sup.4 different proteins present in the human cell. A further electrophoretic analysis system comprised isoelectric focusing which separated proteins in a continuous pH gradient according to differences in isoelectric pH with a maximum resolution at 10.sup.2. Other methods and systems of electrophoresis yielded resolutions of similar magnitude.
It has been suggested that protein mixtures be separated according to both size and charge differences, and a combination of the two procedures has been termed two dimensional electrophoresis. The potential resolution of the two dimensional electrophoresis has been approximated at 5.times.10.sup.2 with the coomassie blue staining method.
Various approaches have been attempted for optimized electrophoretic analysis through two dimensional electrophoresis. In one approach, a sample was first separated by isoelectric focusing within a rod shaped medium which, in turn, was carried in a glass tube. After completing the electrophoresis run, the medium was removed from the tube by mechanical means. Examples of removal techniques included mechanically applying pressure at one end of the tube to extrude the medium at the other end, injecting water between the medium and the tube, alternately freezing and thawing and even breaking the glass tube. In all of such attempts a high risk of damaging the medium was incurred.
The gel extraction procedure presented significant problems affecting reliability. A common supporting matrix in isoelectric focusing was 3% bis-acrylamide which forms a fragile gel. It was extremely difficult to maintain the original shape of the rod shaped medium. Pressure employed during removal, a syringe for injecting water between the rod and the tube or other extraction procedures often deformed or ruptured the rod medium.
After extraction, the separated rod medium was equilibrated to provide a uniform charge to the protein constituents and was placed horizontally on the upper portion of a vertical slab gel. The vertical slab gel was designed for protein separation according to size such as by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Approaches at two dimensional electrophoresis wherein a first run was achieved through a rod which thereafter was removed from a tube were typically illustrated in U.S. Pat. Nos. 4,305,799 and 4,088,561.
Although two dimensional electrophoresis possessed potential for superior diagnostic analysis due to high resolution capabilities, it has not been widely employed. Aside from the difficulty in rod medium removal which was a major disadvantage, the technique was subject to a relatively low degree of reproducibility. This was because isoelectric focusing electrophoresis apparatus and sodium dodecyl sulfate polyacrylamide gel electrophoresis apparatus varied in different laboratories based upon manufacturer and laboratory requirements.
A further problem encountered with prior two dimensional electrophoresis apparatus was that resolution was a function of media thickness. To optimize resolution, the rod media diameter must be minimized and the slab thickness minimized. In an isoelectric focusing run, high resolution could be obtained with a tubing diameter of 1 millimeter and a tubing length of 20 centimeters. While resolution was thus optimized, only a small amount of sample could be separated and only proteins of relatively high concentrations were detected.
It has been proposed to obtain two dimensional electrophoresis without removal of the medium after the first electrophoresis run. U.S. Pat. Nos. 4,101,401 and 4,061,561 are exemplary of such proposals to simplify two dimensional electrophoresis procedures. In U.S. Pat. No. 4,061,561 it was suggested that after an electrophoresis run through a slab medium, the slab be rotated 90 degrees and electrophoresis continue through the same medium in an orthogonal direction. This procedure only provided for a single type of separation.
In U.S. Pat. No. 4,101,401 two dimensional slab gel electrophoresis was suggested with a 90 degrees rotation of the slab. While different mediums were employed for each of the runs, high reliability of results have not been obtained due to a dispersion of the specimen constituents during the initial run. Applicant has determined that optimum results are obtained when the electric field is confined to a definite path such as that defined by a glass tube during the initial run. As a result, attempts at two dimensional electrophoresis as suggested have not provided satisfactory results.